In vitrodegradation of a chitosan-based osteochondral construct points to a transient effect on cellular viability.

Bioresorbable chitosan scaffolds have shown potential for osteochondral repair applications. Thein vivodegradation of chitosan, mediated by lysozyme and releasing glucosamine, enables progressive replacement by ingrowing tissue. Here the degradation process of a chitosan-nHA based bioresorbable scaffold was investigated for mass loss, mechanical properties and degradation products released from the scaffold when subjected to clinically relevant enzyme concentrations. The scaffold showed accelerated mass loss during the early stages of degradation but without substantial reduction in mechanical strength or structure deterioration. Although not cytotoxic, the medium in which the scaffold was degraded for over 2 weeks showed a transient decrease in mesenchymal stem cell viability, and the main degradation product (glucosamine) demonstrated a possible adverse effect on viability when added at its peak concentration. This study has implications for the design and biomedical application of chitosan scaffolds, underlining the importance of modelling degradation products to determine suitability for clinical translation.

Development and in vitro assessment of a bi-layered chitosan-nano-hydroxyapatite osteochondral scaffold.

An innovative approach was developed to engineer a multi-layered chitosan scaffold for osteochondral defect repair. A combination of freeze drying and porogen-leaching out methods produced a porous, bioresorbable scaffold with a distinct gradient of pore size (mean = 160-275 µm). Incorporation of 70 wt% nano-hydroxyapatite (nHA) provided additional strength to the bone-like layer. The scaffold showed instantaneous mechanical recovery under compressive loading and did not delaminate under tensile loading. The scaffold supported the attachment and proliferation of human mesenchymal stem cells (MSCs), with typical adherent cell morphology found on the bone layer compared to a rounded cell morphology on the chondrogenic layer. Osteogenic and chondrogenic differentiation of MSCs preferentially occurred in selected layers of the scaffold in vitro, driven by the distinct pore gradient and material composition. This scaffold is a suitable candidate for minimally invasive arthroscopic delivery in the clinic with potential to regenerate damaged cartilage and bone.

Human osteoblast cell spreading and vinculin expression upon biomaterial surfaces.

Any biomaterial implanted within the human body is influenced by the interactions that take place between its surface and the surrounding biological milieu. These interactions are known to influence the tissue interface dynamic, and thus act to emphasize the need to study cell-surface interactions as part of any biomaterial design process. The work described here investigates the relationship between human osteoblast attachment, spreading and focal contact formation on selected surfaces using immunostaining and digital image processing for vinculin, a key focal adhesion component. Our observations show that a relationship exists between levels of cell attachment, the degree of vinculin-associated plaque formation and biocompatibility. It also suggests that cell adhesion is not indicative of how supportive a substrate is to cell spreading, and that cell spreading does not correlate with focal contact formation.

In vitro degradation and mechanical properties of PLA-PCL copolymer unit cell scaffolds generated by two-photon polymerization.

The manufacture of 3D scaffolds with specific controlled porous architecture, defined microstructure and an adjustable degradation profile was achieved using two-photon polymerization (TPP) with a size of 2 × 4 × 2 mm(3). Scaffolds made from poly(D,L-lactide-co-ɛ-caprolactone) copolymer with varying lactic acid (LA) and ɛ -caprolactone (CL) ratios (LC16:4, 18:2 and 9:1) were generated via ring-opening-polymerization and photoactivation. The reactivity was quantified using photo-DSC, yielding a double bond conversion ranging from 70% to 90%. The pore sizes for all LC scaffolds were see 300 µm and throat sizes varied from 152 to 177 µm. In vitro degradation was conducted at different temperatures; 37, 50 and 65 °C. Change in compressive properties immersed at 37 °C over time was also measured. Variations in thermal, degradation and mechanical properties of the LC scaffolds were related to the LA/CL ratio. Scaffold LC16:4 showed significantly lower glass transition temperature (T g) (4.8 °C) in comparison with the LC 18:2 and 9:1 (see 32 °C). Rates of mass loss for the LC16:4 scaffolds at all temperatures were significantly lower than that for LC18:2 and 9:1. The degradation activation energies for scaffold materials ranged from 82.7 to 94.9 kJ mol(-1). A prediction for degradation time was applied through a correlation between long-term degradation studies at 37 °C and short-term studies at elevated temperatures (50 and 65 °C) using the half-life of mass loss (Time (M1/2)) parameter. However, the initial compressive moduli for LC18:2 and 9:1 scaffolds were 7 to 14 times higher than LC16:4 (see 0.27) which was suggested to be due to its higher CL content (20%). All scaffolds showed a gradual loss in their compressive strength and modulus over time as a result of progressive mass loss over time. The manufacturing process utilized and the scaffolds produced have potential for use in tissue engineering and regenerative medicine applications.

Use of a novel carbon fibre composite material for the femoral stem component of a THR system: in vitro biological assessment.

A novel, low elastic modulus femoral component for THR has been developed using a composite of polyetheretherketone and carbon fibre. The objectives of this study were to investigate human osteoblast-like cell and macrophage responses to this material in vitro. Cells were grown on composite discs and controls. Osteoblast attachment and proliferation was not significantly different to that on Ti6Al4V. The levels of alkaline phosphatase activity, Type I collagen production and osteocalcin production were not significantly different to that on Ti6Al4V by the end of the experimental period. Hydrogen peroxide production by macrophages was significantly less than that detected for cells cultured on copper, but was still greater than that detected for cells cultured on tissue culture plastic and Ti6Al4V. Beta-glucoronidase activity was not significantly different to that detected for cells cultured on tissue culture plastic. The in vitro biocompatibility assessment of this composite undertaken in this study showed initial osteoblast attachment at least comparable to that of the tissue culture plastic and Ti6Al4V controls, with proliferation similar to the controls at all time points up to 11 days. Alkaline phosphatase activity was similar to that of Ti6Al4V but reduced compared to tissue culture plastic controls. Whilst hydrogen peroxide production by macrophages was raised on composite surfaces compared to controls, beta-glucoronidase activity and osteoblastic production of Type I collagen and osteocalcin were similar to levels detected on Ti6Al4V.

Comparison of environmental scanning electron microscopy with high vacuum scanning electron microscopy as applied to the assessment of cell morphology.

The efficacy of conventional high vacuum scanning electron microscopy (SEM), environmental SEM (ESEM), and confocal laser scanning microscopy techniques in the assessment of cell-material interactions is compared. Specific attention is given to the application of these techniques in the assessment of the early morphological response of human osteoblast-like cells cultured on titanium dioxide. The processing of cells cultured for conventional high vacuum SEM leads to the loss of morphological features that are retained when using ESEM. The use of cytoskeletal labeling, viewed with confocal laser scanning microscopy, in conjunction with ESEM gives an indication of the changes to cell morphology as a consequence of incubation time in response to interactions at the biological/material interface.

Development of an elastic cell culture substrate for a novel uniaxial tensile strain bioreactor.

Bioreactors can be used for mechanical conditioning and to investigate the mechanobiology of cells in vitro. In this study a polyurethane (PU), Chronoflex AL, was evaluated for use as a flexible cell culture substrate in a novel bioreactor capable of imparting cyclic uniaxial tensile strain to cells. PU membranes were plasma etched, across a range of operating parameters, in oxygen. Contact angle analysis and X-ray photoelectron spectroscopy showed increases in wettability and surface oxygen were related to both etching power and duration. Atomic force microscopy demonstrated that surface roughness decreased after etching at 20 W but was increased at higher powers. The etching parameters, 20 W 40 s, produced membranes with high surface oxygen content (21%), a contact angle of 66° ± 7° and reduced topographical features. Etching and protein conditioning membranes facilitated attachment, and growth to confluence within 3 days, of MG-63 osteoblasts. After 2 days with uniaxial strain (1%, 30 cycles/min, 1500 cycles/day), cellular alignment was observed perpendicular to the principal strain axis, and found to increase after 24 h. The results indicate that the membrane supports culture and strain transmission to adhered cells.

Topographical and chemical effects of electrochemically assisted deposited hydroxyapatite coatings on osteoblast-like cells.

A recently commercialised hydroxyapatite electrochemically assisted chemical deposition technique (BoneMaster) has been shown to induce increased bone apposition; whether this response is caused by the surface topography or chemistry is unknown. An in-vitro examination using human osteoblast-like cells was performed on a series of BoneMaster-coated surfaces. The chemistry was separated from the topography using a thin gold coating; Thermanox coverslips were used as a control. BoneMaster surfaces showed significantly greater alkaline phosphatase activity and osteocalcin production compared with controls; however, no difference was found between the gold-coated and uncoated BoneMaster samples, indicating topography is the main contributing factor.

Composite PLA scaffolds reinforced with PDO fibers for tissue engineering.

Novel composite scaffolds were produced using long continuous bidirectional fibers embedded in an electrospun matrix, with the aim of using them in soft tissue engineering applications. The fibers are of polydioxanone and the matrix of polylactic acid. The novel manufacturing method consists of direct electrospinning performed on both sides of a collector that supports the already arranged fibers. The scaffolds were tested in vitro using 3T3 mouse fibroblasts as-obtained or functionalized with biotin or poly (dopamine). Functionalization did not significantly affect cells attachment, metabolic activity, or proliferation, but poly (dopamine) was proven to be effective in inducing hydrophilicity to the surface.

Effect of boron addition on the thermal, degradation, and cytocompatibility properties of phosphate-based glasses.

In this study eight different phosphate-based glass compositions were prepared by melt-quenching: four in the (P2O5)45-(CaO)16-(Na2O)15-x -(MgO)24-(B2O3) x system and four in the system (P2O5)50-(CaO)16-(Na2O)10-x -(MgO)24-(B2O3) x , where x = 0,1, 5 and 10 mol%. The effect of B2O3 addition on the thermal properties, density, molar volume, dissolution rates, and cytocompatibility were studied for both glass systems. Addition of B2O3 increased the glass transition (T(g)), crystallisation (T(c)), melting (T(m)), Liquidus (T(L)) and dilatometric softening (T(d)) temperature and molar volume (V(m)). The thermal expansion coefficient (α) and density (ρ) were seen to decrease. An assessment of the thermal stability of the glasses was made in terms of their processing window (crystallisation onset, T(c,ons) minus glass transition temperature, T(g)), and an increase in the processing window was observed with increasing B2O3 content. Degradation studies of the glasses revealed that the rates decreased with increasing B2O3 content and a decrease in degradation rates was also observed as the P2O5 content reduced from 50 to 45 mol%. MG63 osteoblast-like cells cultured in direct contact with the glass samples for 14 days revealed comparative data to the positive control for the cell metabolic activity, proliferation, ALP activity, and morphology for glasses containing up to 5 mol% of B2O3.

Influence of screw holes and gamma sterilization on properties of phosphate glass fiber-reinforced composite bone plates.

Polymers prepared from polylactic acid (PLA) have found a multitude of uses as medical devices. For a material that degrades, the main advantage is that an implant would not necessitate a second surgical event for removal. In this study, fibers produced from a quaternary phosphate-based glass (PBG) in the system 50P2O5-40CaO-5Na2O-5Fe2O3 were used to reinforce PLA polymer. The purpose of this study was to assess the effect of screw holes in a range of PBG-reinforced PLA composites with varying fiber layup and volume fraction. The flexural properties obtained showed that the strength and modulus values increased with increasing fiber volume fraction; from 96 MPa to 320 MPa for strength and between 4 GPa and 24 GPa for modulus. Furthermore, utilizing a larger number of thinner unidirectional (UD) fiber prepreg layers provided a significant increase in mechanical properties, which was attributed to enhanced wet out and thus better fiber dispersion during production. The effect of gamma sterilization via flexural tests showed no statistically significant difference between the sterilized and nonsterilized samples, with the exception of the modulus values for samples with screw holes. Degradation profiles revealed that samples with screw holes degraded faster than those without screw holes due to an increased surface area for the plates with screw holes in PBS up to 30 days. Scanning electron microscope (SEM) analysis revealed fiber pullout before and after degradation. Compared with various fiber impregnation samples, with 25% volume fraction, 8 thinner unidirectional prepreg stacked samples had the shortest fiber pull-out lengths in comparison to the other samples investigated.

Investigating the use of coupling agents to improve the interfacial properties between a resorbable phosphate glass and polylactic acid matrix.

Eight different chemicals were investigated as potential candidate coupling agents for phosphate glass fibre reinforced polylactic acid composites. Evidence of reaction of the coupling agents with phosphate glass and their effect on surface wettability and glass degradation were studied along with their principle role of improving the interface between glass reinforcement and polymer matrix. It was found that, with an optimal amount of coupling agent on the surface of the glass/polymer, interfacial shear strength improved by a factor of 5. Evidence of covalent bonding between agent and glass was found for three of the coupling agents investigated, namely: 3-aminopropyltriethoxysilane; etidronic acid and hexamethylene diisocyanate. These three coupling agents also improved the interfacial shear strength and increased the hydrophobicity of the glass surface. It is expected that this would provide an improvement in the macroscopic properties of full-scale composites fabricated from the same materials which may also help to retain these properties for the desired length of time by retarding the breakdown of the fibre/matrix interface within these composites.

Cytocompatibility and effect of increasing MgO content in a range of quaternary invert phosphate-based glasses.

Recently, phosphate-based glass (PBG) fibers have been used to reinforce the biodegradable polymers polycaprolactone and polylactic acid, in order to fabricate materials suitable for use as resorbable bone fracture fixation devices. However, the PBG fibers investigated tended to degrade too quickly for application. Therefore, more durable PBG formulations were sought with emphasis remaining firmly placed on their biocompatibility. In this study, four invert PBG formulations (in the system P2O5-CaO-MgO-Na2O) were produced with fixed phosphate and calcium content at 40 and 25 mol%, respectively. MgO was added at 10-30 mol% in place of Na2O and the maximum divalent cation to phosphate ratio obtained was 1.375. Thermal analyses showed a linear increase in T(g) with increasing MgO content. This was proposed to be due to an increase in the cross-link density of the glass network, which also improved the chemical durability of the glass. EDX analyses were also conducted to verify the final composition of the glass. XRD analyses confirmed the amorphous nature of the glasses investigated. Rapid quenching of the Mg30 glass revealed a degree of surface crystallization, which was shown to be a CaMgP2O7 phase. The degradation rates of the glasses investigated decreased with increasing MgO content. The decrease in rate seen was almost two orders of magnitude (a x 50 difference was seen between glass Mg0 and Mg30). The cytocompatibility studies of the formulations investigated showed good cellular response over time for up to 14 days. Statistical analysis revealed that the formulations investigated gave a response comparable to the tissue culture plastic control. It is suggested that invert PBG provide degradation profiles and the cytocompatibility response desired to make these glasses useful for bone repair applications.

Mono-functional aminosilanes as primers for peptide functionalization.

When covalently attaching biomolecules to surfaces such as titanium, trifunctional silanes are commonly used as primers to produce surface amine groups. However, these primed surfaces are rarely uniform in structure due to networking of the silane. Mono-functional aminosilanes may result in more uniform structures, although their long-term stability and effect on osteoblast cell responses are possible issues for orthopedic applications. This study examines for the first time the optimization of peptide coupling to titanium using mono-functional aminosilane reaction chemistry. The resultant surface topography, chemistry, and thicknesses were characterized showing improved surface uniformity compared with trifunctional silanized surfaces. The stability of the coatings was examined over a period of 8 days in environments of varying pH, temperature, and humidity. In addition, human osteosarcoma (HOS) cell adhesion and spreading on the samples was examined; adhesion was minimal on silanized surfaces, but after functionalization with cysteine the cell density was greater than the titanium control and showed no overall detrimental effect on initial cell responses.

Analysis of calvarial bone defects in rats using microcomputed tomography: potential for a novel composite material and a new quantitative measurement.

Reconstruction of craniomaxillofacial defects is a challenge for surgeons and has psychological and functional burdens for patients. Undoubtedly, there is a need for improved biomaterials and techniques for craniomaxillofacial reconstruction. We assessed the potential regeneration of bone using three modifications of a novel composite and explored the validity of a new measurement using microcomputed tomography (micro-CT). We placed three different composite samples in calvarial defects in rats and analysed healing with micro-CT. The results showed that polycaprolactone (PCL) with phosphate glass fibre is promising for non-load bearing applications in the craniomaxillofacial region. Also, the new micro-CT measurement of the temporal characterisation of the mineralisation of bone (TCBM) has the potential to evolve into a reliable predictor of bony healing and its quality.

The effect of different surface morphology and roughness on osteoblast-like cells.

Increased magnitude of biomaterial surface roughness and micromachined-grooved surfaces has both been shown to stimulate osteoblast activity, but have not been compared in the same study quantitatively. A series of titanium alloy (Ti6Al4V) samples were prepared using simple machining techniques to undertake such a comparison. Samples were either grit blasted (Gb) or shot peened (Sp) to give random discontinuities, or silicon carbide ground (SiC) to produce ordered grooves. These were compared with micropolished samples (Mp). The samples were coated with a 1 mum continuous coating of hydroxyapatite to remove differences in surface chemistry. Human osteoblast-like cells were seeded onto the materials and metabolic activity, proliferation, alkaline phosphatase activity, and osteocalcin production assessed. Cell responses were highly dependent on the substrate that they were cultured on. Cells cultured on the smooth and ordered (Mp and SiC, respectively) samples had higher metabolic activity and a more elongated morphology than those cultured on the randomly structured Gb or Sp samples. Over 21 days, cell metabolic activity peaked relative to the control between 7 and 14 days on the Mp sample, and between 14 and 21 days on the Gb, Sp, and SiC samples. In common with other researchers, we note that micron scale topography may have potential for influencing osseointegration. More importantly, as the magnitude of the discontinuities on SiC, Gb, and Sp were similar, the differences in cell responses does not appear to lie with the size of the features, but whether the features showed an ordered or disordered structure.

In vitro study of hydroxyapatite-based photocurable polymer composites prepared by laser stereolithography and supercritical fluid extraction.

The fabrication of three-dimensional (3-D) structures using computer-controlled ultraviolet (UV) photopolymerization of acrylates (laser stereolithography) often results in the trapping of residual unreacted monomer and initiator. These residuals can leach from the finished structure and affect the biological response of cells and tissues. Thus the potential applications of these structures for tissue engineering have not been fully realized. In this paper we demonstrate that conventional post-lithography treatments followed by processing in the environmentally benign solvent, supercritical carbon dioxide (scCO(2)), dramatically increased biocompatibility. The scCO(2) processing of pure polyacrylate and polyacrylate/hydroxyapatite composite structures extracts residuals from all structures including those that had received full conventional post-lithography treatment (acetone washing/UV drying). Human osteoblast cells seeded on the extracted surfaces of these structures demonstrated increased cell attachment and proliferation on the scCO(2)-treated materials.

Cytotoxicity of glutaraldehyde crosslinked collagen/poly(vinyl alcohol) films is by the mechanism of apoptosis.

Collagen has been investigated as a potential natural biomaterial, because of its occurrence in the extracellular matrix. Collagen requires crosslinking in this context, by reagents that are often cytotoxic. Glutaraldehyde is one such agent that is potentially cytotoxic. The aim of this study was to determine the cause of poor cell attachment and growth on collagen/poly(vinyl alcohol) bioartificial composite films, when crosslinked with glutaraldehyde. Dehydrothermal crosslinking was used as a comparison. Human osteoblasts were observed to undergo apoptosis on glutaraldehyde crosslinked films dependent on concentration of collagen present. Higher collagen content resulted in higher levels of apoptosis with poor cell attachment and spreading of remaining cells. Post-treatment of films with 8% L-glutamic acid prevented the apoptotic response of osteoblasts and allowed attachment and spreading. The addition of 100 nM insulin-like growth factor-1 to the culture medium also prevented apoptosis. Glutaraldehyde toxicity of crosslinked collagen has been demonstrated in this study, the mechanism of which is apoptosis. This study indicates that poor biocompatibility and induction of apoptosis on collagen/poly(vinyl alcohol) films crosslinked by glutaraldehyde are attributed to glutaraldehyde components on the surface of the films (not residual glutaraldehyde), whose effects can be quenched by glutamic acid, and prevented by insulin-like growth factor-1.